DEV012856 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Procedures for cell and bead implantations into presumptive gonadal region. (A) Procedure for cell implantation. (a) Chick embryos at stage 11-12 were used. Somites (so) are developing from anterior to posterior. Although the somites at the level of the presumptive gonadal region are not developed yet at this stage, the presumptive regions for forelimb (15-20 somites), gonad (21-25 somites) and hindlimb (26-32 somites) were predicted as indicated. (b) A sharp tungsten needle was used to cut a slit into the presumptive gonadal region of lateral plate mesoderm (lpm) at the level of 22-24 somites, and the cells producing recombinant viruses were implanted through the slit (red arrows). (B) Procedure for bead implantation. (a) Chick embryos at stage 18-19 were used. A sharp tungsten needle was used to cut a slit adjacent to the embryonic body trunk at the level of 22-24 somites. As indicated by red arrows, beads were introduced into the visceral cavity (vc) through the slit. (b,c) The beads were placed at the presumptive gonad region (pgr) using a glass needle. Spm, segmental plate mesoderm; nt, neural tube; nd, nephric duct.

• Supplemental Figure S2 -

  Fig. S2. L-R asymmetric morphology and symmetric apoptosis in developing gonads. (A-D) Right (A) and left (B) gonads of a female embryo (ZW) at stage 27. Right (C) and left (D) gonads of a female embryo (ZW) at stage 29 were fixed in 3% glutaraldehyde in 10 mM HEPES-buffered saline (pH 7.4) for at least 2 hours. After post-fixation with 1% osmium tetroxide for 1 hour, the tissues were dehydrated and embedded in epoxy resin. Semi-thin sections (1.5 µm) were prepared and stained with 0.5% Toluidine Blue. Regions surrounded by rectangles in C and D are enlarged in insets. Borders between the cortex and medulla are indicated by dotted lines, with m and c indicating the medulla and cortex, respectively. (E-H) TUNEL assays were performed with an in situ apoptosis detection kit (Chemicon International) on gonad sections at stage 27 and 29. The sections were incubated with sheep anti-digoxigenin polyclonal antibody (green) and mouse anti-cytokeratin monoclonal antibody (red) for 1 hour at 37°C. Alexa Fluor 488-labelled donkey anti-sheep antibody (1:200; Molecular Probes) and Alexa Fluor 555-labelled goat anti-mouse antibody (1:500; Molecular Probes) were used as secondary antibodies. Total cells were stained with DAPI. Overlaid images of DAPI and cytokeratin (E,F), and TUNEL and cytokeratin (G,H), staining are shown. Male (ZZ) embryos were also used for these studies, and similar observations were obtained. R and L indicate right and left. Scale bars: 50 µm in A-D; 25 µm in inset in C and D; 100 µm in E-H.

• Supplemental Figure S3 -

  Fig. S3. Luciferase reporter expression driven by wild-type and dominant-negative Pitx2. (A) A cDNA encoding mouse Pitx2c amino acids 35-188 was cloned into pCR II-TOPO vector (Invitrogen). To construct the dominant-negative Pitx2-en, the repressor domain of Drosophila engrailed (amino acids 1-296) (Jaynes and O'Farrell, 1991) was fused to the carboxy-terminus of Pitx2c. Pitx2 and Pitx2-en were subcloned into the expression vector pCMX (Umesono et al., 1991). As the reporter gene, five tandem repeats of an oligonucleotide containing the Pitx2 binding site (underlined) (5′-AGCTTGCACGGCCCATCTAATCCCGTGT-3′) were placed upstream of the thymidine kinase basal promoter and a luciferase reporter gene. (B) Transcriptional activities of mouse Pitx2 and Pitx2-En were analyzed in transient transfection assays. 6×10⁴ CV-1 cells were seeded in 24-well plates 24 hours before transfection with lipofectamine reagent (Invitrogen). The total amount of transfected plasmid (including 50 ng luciferase reporter plasmid) was adjusted to 550 ng with empty expression vector. Cells were harvested 36 hours after transfection and cell lysates were assayed for luciferase activity (Mukai et al., 2002). All transfection experiments were performed in triplicate.

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